The immune response comprises a cellular response and a humoral response. The cellular response is mediated largely by T-lymphocytes (alternatively and equivalently referred to herein as T cells), while the humoral response is mediated by B-lymphocytes (alternatively and equivalently referred to herein as B-cells).
B-cells produce and secrete antibodies in response to the presentation of antigen and MHC class II molecules on the surface of antigen presenting cells. Antigen presentation initiates B-cell activation with the engagement of the B-cell receptor (BCR) at the cell's surface. Following engagement, the BCR relays signals that are propagated through the cell's interior via signal transduction pathways. These signals lead to changes in B-cell gene expression and physiology, which underlie B-cell activation.
T cells produce costimulatory molecules, including soluble cytokines and cell surface proteins, that augment antibody production by B-cells during the humoral immune response. Cytokines also play a role in modulating the activity of T cells themselves. Many T cells act directly to engulf and destroy cells or agents that they recognize by virtue of the cell surface proteins they possess. The engagement of cell surface receptors on T cells results in the propagation of intracellular signals that provoke changes in T cell gene expression and physiology, which underlie the cellular immune response. B-cells in turn express cell surface proteins that modulate the activity of T cells and cellular immunity.
Antigen recognition alone is usually not sufficient to initiate a complete effector T or B cell response. The generation of many B-cell responses to antigen is dependent upon the interaction of B-cells with CD4+ helper T cells directed against the same antigen. These helper T cells express the CD40 ligand “CD40L” (also known as CD154) which binds to the cell surface receptor, CD40, on resting B-cells. This interaction provides a critical activation signal to B-cells. Mutations in the CD40L lead to the X-linked immunodeficiency disorder hyper-IgM syndrome, which is characterized by low levels of IgA and IgG, normal to elevated levels of IgM, absence of germinal center formation, and decreased immune response. In addition, transgenic mice lacking CD40 exhibit reduced graft rejection. (Zanelli et al., Nature Medicine, 6: 629-630, 2000; Schonbeck et al., Cell Mol Life Sci, 58:4-43, 2001).
CD40L expression on helper T cells is in turn regulated by the expression of CD80 on B cells. CD80 interacts with the T cell surface receptors CD28 and CD154 to modulate the activity of T cells, including the B cell-modulating activity of T cells (Sperling et al., 153:155-182, 1996). CD80 expression is in turn regulated by CD40 activity in B cells.
CD80 is expressed in a variety of antigen presenting cells (APCs) including B cells and dendritic cells and is essential for T cell co-stimulation. When T cells encounter peptide-MHC complexes on an APC, CD28 acts in conjunction with the TCR to produce a maximal cellular response. CD80 is currently being evaluated as a target for the treatment of clinical conditions including transplantation, graft-versus-host disease, psoriasis, and rheumatoid arthritis.
Non-lymphocyte myeloid derivatives are also activated by surface receptor engagement in immune response and in response to injury. For example, mast cells and basophils are activated by binding of antigen to surface IgE, while platelets are activated by the binding of thrombin to its receptor.
Intercellular communication between different types of lymphocytes, as well as between lymphocytes and non-lymphocytes in the normally functioning immune system is well known. Much of this communication is mediated by cytokines and their cognate receptors. Cytokine-induced signals begin at the cell surface with a cytokine receptor and are transmitted intracellularly via signal transduction pathways. Many types of cells produce cytokines, and cytokines can induce a variety of responses in a variety of cell types, including lymphocytes. The response to a cytokine can be context-dependent as well as cell type-specific.
In addition, the expression of cytokine receptor subunits may be dynamically regulated. For example, the II-2 receptor α-subunit (CD25) is dynamically regulated in B cells and expression has been associated with leukemia (de Totero et al., Leukemia 9:1425-1431, 1995; Leonard et al., Cold Spring Harbor Symp. Quant. Biol., 64:417-424, 1999).
Dysregulation of intercellular communication can perturb lymphocyte activation and the regulation of immune responses. Such dysregulation is believed to underlie certain autoimmune disease states, hyper-immune states, and immune-compromised states. Such dysfunction may be cell autonomous or non-cell autonomous with respect to lymphocytes.
The activation of specific signaling pathways in lymphocytes determines the quality, magnitude, and duration of immune responses. In response to transplantation, in acute and chronic inflammatory diseases, and in autoimmune responses, it is these pathways that are responsible for the induction, maintenance and exacerbation of undesirable lymphocyte responses. Identification of these signaling pathways is desirable in order to provide diagnostic and prognostic tools, as well as therapeutic targets for modulating lymphocyte function in a variety of disorders or abnormal physiological states. In addition, the ability to modulate these pathways and suppress normal immune responses is often desirable, for example in the treatment of hosts receiving a transplant.
While the extracellular domains and cognate ligands of lymphocyte receptors vary widely, many receptors have similar intracellular domains (such as the “immunoreceptor tyrosine-based activation motif” (ITAM)), and associate with common intracellular signaling molecules.
Tyrosine kinase activation is a critical event in the propagation of intracellular signals by many receptors on lymphocytes, including antigen receptors on B and T cells (for a review see Turner et al, Immunology Today, 21:148-154, 2000, incorporated herein in its entirety by reference).
With regard to the B-cell antigen receptor (BCR), the BCR is rapidly phosphorylated on tyrosine residues following engagement of the receptor by antigen or other crosslinking agents. This tyrosine phosphorylation leads to associations with several SH2-containing signaling proteins. SH2-containing proteins are known to bind to phosphorylated tyrosine residues in the context of specific amino acid sequences (Pawson, Nature, 373:573-580, 1995; Pawson et al., Science, 278:2075-2080, 1997).
Many non-receptor tyrosine kinases have been shown to interact with tyrosine phosphorylated receptors in lymphocytes, including the antigen receptors of B and T cells. These non-receptor tyrosine kinases include members of the src family and the Bruton's tyrosine kinase (BTK) family. Importantly, many of these genes are associated with oncogenesis (van Leeuwen et al., Curr. Opin. Immunol., 11:242-248, 1999).
Btk is a non-receptor tyrosine kinase that is involved in the BCR signaling pathway and is critical for B cell development (for a review, see Tsukada et al., Advances in Immunology, 77:123-162, 2001; incorporated herein by reference). Btk was originally identified as a tyrosine kinase deficiency in human X-linked agammaglobulinemia. The Btk protein comprises a number of motifs, including SH1, SH2, SH3 and PH motifs, and interacts with a number of proteins in the BCR signal transduction pathway.
The present application discloses the finding that Btk binds to Mkk3b, a dual specificity kinase.
Mkk3b is a known dual specificity kinase that phosphorylates and activates p38 MAP kinase on specific threonine and tyrosine residues (Han et al., FEBS Letters 403:19-22, 1997; Ravanti et al., J. Biol. Chem. 274:37292-300, 1999). A second isoform, namely Mkk3, has similar function but is much less efficient at phosphorylating and activating p38 MAP kinase (Han et al., ibid). Mkk3b has an additional 29 amino acids at the N-terminus compared with Mkk3. Both Mkk3 and Mkk3b comprise a kinase domain, which can be further subdivided into an ATP pocket and an activation site. The activation site contains a serine at position 218, and a threonine at position 222.
Mkk3 responds to a variety of stresses such as shear stree, oxidative stress, and osmotic shock, as well as to cytokines and growth factors such as TNF, and FGF (Han et al., supra; Wysk et al., Proc. Natl Acad. Sci., 96:3763-3768, 1999; Matsumoto et al., J. Cell Biol., 156:149-160, 2002; Davis et. al., U.S. Pat. No. 5,736,381; Davis et. al., U.S. Pat. No. 5,804,427; Davis et. al. WO 96/36642). Mkk3b is activated by phosphorylation of residues serine 218 (S218) and threonine 222 (T222). Mkk3 is activated by mixed lineage kinase 1 (MLK3) (Tibbles et al., EMBO J, 15:7026-7035,1996), and apoptosis signal-regulating kinase 1 (ASK1) (Ichijo et al., Science, 275:90-94, 1997). Autophosphorylation of Mkk3 has been detected (Derijard et al., Science 267:682-685,1995) and Mkk3b is dephosphorylated by protein phosphatase 2C (Hanada et al, FEBS Letters 437:172-176, 1998). A constitutively active variant of Mkk3b which does not require phosphorylation in order to exhibit Mkk3b bioactivity has also been generated (Wang et al., J. Biol. Chem., 273:2161-2168, 1998).
Once activated, Mkk3b phosphorylates p38, a kinase involved in the inflammatory responses including the regulation of T and B cell activation and differentiation. The activation of human p38 is mediated by phosphorylation of threonine residue 180 and tyrosine residue 182 by Mkk3 and Mkk3b (Derijard et al., Science 267:682-685, 1995; Han et al., FEBS Letters 403:19-22, 1997; Moriguchi et al., J. Biol. Chem. 271:26981-8, 1996). Homozygous p38 gene inactivation in mice is lethal. Most p38 nullizygous mice die during embryogenesis; those that survive exhibit a deficit in erythropoietin expression and die early in adulthood (Tamura et al., Cell 102:221-231, 2000).
In contrast, mice deficient in Mkk3 function exhibit inflammatory, but not systemic, defects. Inactivation of the Mkk3 gene in mice has been shown to perturb the Th1 response, and Mkk3 deficient macrophages and antigen presenting cells exhibit a deficit in IL-12 induction in response stimulation with LPS or CD40L. Mkk3 nullizygous mice also exhibit a defect in interferon-gamma (IFN-γ) secretion by Th1 cells, and in proinflammatory cytokine production by fibroblasts following TNF stimulation (Lu et al., EMBO J. 18:1845-1857, 1999). Importantly, Mkk3-deficient mice are viable and fertile, exhibit no developmental defects, have no gross histological abnormalities, and exhibit normal numbers and development of B cells, T cells, monocytes, and dendritic cells.
Despite its implication in inflammatory processes, a role for Mkk3b in B cells has not been established.